Repairing compound heterozygous recessive mutations by allele exchange

Abstract

The disclosure in some aspects relates to methods and compositions for repairing mutations (e.g., compound heterozygous mutations) that are widely found in patients having certain diseases (e.g., monogenic recessive diseases). In some aspects, the disclosure provides a method for targeted allelic exchange using recombinant gene editing complex.

What is claimed is: 
     
       1. A method comprising contacting a pair of homologous chromosomes, each chromosome of the pair having a heterogeneous recessive allele of the same gene, each heterogeneous recessive allele having a positionally unique mutation, with a recombinant gene editing complex, under conditions under which the recombinant gene editing complex induces double stranded breaks in each of the two chromosomes at an intronic site aligning between the positionally unique mutations to produce a mutation-free chromosome and a mutant chromosome via non-homologous end-joining (NHEJ); wherein the recombinant gene editing complex comprises a Cas protein, and a guide RNA (gRNA) or single-stranded guide RNA (sgRNA) that is complementary to SEQ ID NOs: 6 or 7. 
     
     
       2. The method of  claim 1 , wherein the position of each heterogeneous recessive allele is separated by at least one complete intron. 
     
     
       3. The method of  claim 2 , wherein the complete intron is at least 50 nucleotides in length. 
     
     
       4. The method of  claim 1 , wherein each heterogeneous allele comprises a polymorphism selected from a single nucleotide polymorphism (SNP), an insertion polymorphism, or a deletion polymorphism. 
     
     
       5. The method of  claim 4 , wherein the polymorphism is associated with a disease. 
     
     
       6. The method of  claim 5 , wherein the disease is selected from the group consisting of tyrosinemia and Canavan disease. 
     
     
       7. The method of  claim 1 , wherein the Cas protein is a Cas9 protein or a Cpf1 protein. 
     
     
       8. The method of  claim 1 , wherein the method comprises contacting the pair of homologous chromosomes in a cell. 
     
     
       9. A method comprising delivering to a cell at least one component of a recombinant gene-editing complex, the cell having a pair of homologous chromosomes, each chromosome of the pair having a heterogeneous recessive allele of the same gene, each heterogeneous recessive allele having a positionally unique mutation, with a recombinant gene editing complex, under conditions under which the recombinant gene editing complex induces double stranded breaks in each of the two chromosomes at an intronic site aligning between the positionally unique mutations to produce a mutation-free chromosome and a mutant chromosome via non-homologous end-joining (NHEJ); wherein the recombinant gene-editing complex comprises a Cas protein, and a guide RNA (gRNA) or single-stranded guide RNA (sgRNA); and wherein the gRNA or sgRNA is complementary to SEQ ID NOs: 6 or 7. 
     
     
       10. The method of  claim 9 , wherein the cell is in a subject. 
     
     
       11. The method of  claim 9 , wherein the cell is in vitro or ex vivo. 
     
     
       12. The method of  claim 9 , wherein the at least one component of the gene editing complex is delivered to the cell in a recombinant adeno-associated virus (rAAV). 
     
     
       13. A method comprising administering to a subject at least one component of a recombinant gene-editing complex, the subject having a cell comprising a pair of homologous chromosomes, each chromosome of the pair having a heterogeneous recessive allele of the same gene, each heterogeneous recessive allele having a positionally unique mutation, with a recombinant gene editing complex, under conditions under which the recombinant gene editing complex induces double stranded breaks in each of the two chromosomes at an intronic site aligning between the positionally unique mutations, wherein the administered gene-editing complex enters the cell and produces a mutation-free chromosome and a mutant chromosome in the cell via non-homologous end-joining (NHEJ); wherein the recombinant gene-editing complex comprises a Cas protein, and a guide RNA (gRNA) or single-stranded guide RNA (sgRNA); and wherein the gRNA or sgRNA is complementary to SEQ ID NOs: 6 or 7. 
     
     
       14. The method of  claim 13 , wherein the at least one component of the recombinant gene editing complex is administered to the subject by injection. 
     
     
       15. The method of  claim 13 , wherein the subject is a mammal.